The Construction Of Apple MdMYB4 Genes Expression Vector,Callus Transformation And Salt Tolerance Characterization

Apple is one of major fruits in our country,which has rich germplasm resources.However,in the growth process,it will be subjected to various abiotic stresses,such as drought,salinity,high/low temperature and oxidative stress,leading to environmental degradation,so it seriously affect the agricultural production.The plants will be respond to the various stresses,transmitting the stress signal,taking part in the physiological and biochemical process,which can enhance the adaptation to the environment.Abiotic stress has seriously affected the growth and development of plants,besides,the MYB transcription factors play an important role in answering abiotic stresses.To date,the MYB transcription factors have a comparative study in Arabidopsis and tobacco while rarely in woody plants,especially in important crops of apple.In this study,we took the,,Orin’ callus as the research material,cloned the apple gene MdMYB4,analyzed the bioinformatics,and obtained the transgenic callus of MdMYB4.Then we determined the expression level of MdMYB4 and its related genes MdNHX1 and MdSOS1 under different concentration of NaCl using qRT-PCR technology,and obtained recombinant protein by prokaryotic expression.It would lay a foundation for further research of the function of MdMYB4.The main results are as follows:(1)We obtained the full-length cDNA of the salt resistance gene MdMYB4(gene sequence number:MDP0000230141).The completed ORF was 813bp and encoded 270 amino acids,Amino acid composition is 5.25.(2)MdMYB4 from apple and 5 other MYB homologous genes including VvMYB13,PbMYB13,AtMYB13,AtMYB14 and AtMYB15 were used for protein sequence alignment.The protein sequence alignment revealed that the R2R3 domains of MYB TFs were conserved at the N-terminus,but their C-terminal sequences were diverse.All of these genes contained a conserved SG2 motif(MxFW[F/Y]D);(3)Subcellular localization of MdMYB4 in onion epidermal cells was identified.The result showed that MdMYB4 was mainly expressed in the nucleus.(4)Under different concentration of NaCl,overexpressing of MdMYB4 in,,Orin’ callus growed better than WT,indicating that the MdMYB4 was induced by salt tolerance.The results showed that transgenic callus of MdMYB4 could enhance the salt tolerance of,,Orin’callus.(5)The results of qRT-PCR showed that overexpressing of MdMYB4 in,,Orin’ callus,the expression level of MdNHXl was consistent with MdMYB4,and analyzing the MdNHXl promoter showed that it contained the MYB binding sites.So MdMYB4 of R2R3-MYB TFs could regulate the expression of MdNHXl.